Wastewater-based sequencing of Respiratory Syncytial Virus enables tracking of lineages and identifying mutations at antigenic sites

Respiratory Syncytial Virus (RSV) infections pose a substantial health burden, especially for vulnerable populations such as infants and the elderly. While novel immunoprophylaxis offer promising protection, many countries lack robust surveillance systems to monitor circulating RSV lineages and detect mutations that might compromise the effectiveness of these new interventions. In this study, we applied an RSV subtype-specific amplicon-based sequencing approach to obtain RSV-A and RSV-B sequences from wastewater, providing catchment-wide insights into circulating RSV lineages. RSV was amplified and sequenced from wastewater samples collected during the 2022-2023 and 2023-2024 RSV seasons from wastewater treatment plants in Zurich and Geneva, Switzerland. During the 2022-2023 season, RSV-B was the predominant subtype, with the B.D.E.1 lineage prevailing in both cities. In the 2023-2024 season, RSV-A predominated and wastewater sequence data revealed co-circulation of multiple lineages, including A.D.1, A.D.3, A.D.5, and their sub-lineages. Wastewater-derived fusion gene sequences revealed several amino acid substitutions compared to the reference genomes. Most of these corresponded to known signatures of circulating lineages. However, low-frequency, non-synonymous mutations in antigenic sites of both RSV-A and RSV-B were also identified, of which some had not been reported in clinical sequences. These findings demonstrate the potential of wastewater-based genomic surveillance to identify and track circulating RSV lineages and clinically relevant mutations. As novel RSV immunoprophylaxis measures are introduced in upcoming RSV seasons, wastewater-derived genomic RSV data provides a valuable baseline for understanding RSV diversity and future viral evolution under immunological pressure.